A systematic review on the contribution of DNA methylation to hearing loss

Background DNA methylation may have a regulatory role in monogenic sensorineural hearing loss and complex, polygenic phenotypic forms of hearing loss, including age-related hearing impairment or Meniere disease. The purpose of this systematic review is to critically assess the evidence supporting a functional role of DNA methylation in phenotypes associated with hearing loss. Results The search strategy yielded a total of 661 articles. After quality assessment, 25 records were selected (12 human DNA methylation studies, 5 experimental animal studies and 8 studies reporting mutations in the DNMT1 gene). Although some methylation studies reported significant differences in CpG methylation in diverse gene promoters associated with complex hearing loss phenotypes (ARHI, otosclerosis, MD), only one study included a replication cohort that supported a regulatory role for CpG methylation in the genes TCF25 and POLE in ARHI. Conversely, several studies have independently confirmed pathogenic mutations within exon 21 of the DNMT1 gene, which encodes the DNA (cytosine-5)-methyltransferase 1 enzyme. This methylation enzyme is strongly associated with a rare disease defined by autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). Of note, rare variants in DNMT1 and DNMT3A genes have also been reported in noise-induced hearing loss. Conclusions Evidence supporting a functional role for DNA methylation in hearing loss is limited to few genes in complex disorders such as ARHI. Mutations in the DNMT1 gene are associated with ADCA-DN, suggesting the CpG methylation in hearing loss genes deserves further attention in hearing research. Supplementary Information The online version contains supplementary material available at 10.1186/s13148-024-01697-9.


Introduction
Hearing loss in humans is one of the major burdens of disease worldwide [1].Sensorineural hearing loss (SNHL) is the most common type, and it results from abnormal sound processing in the organ of Corti, the auditory pathway or auditory cortex.According to its etiology, SNHL is classified as genetic SNHL and acquired SNHL.Most non-syndromic genetic deafness are monogenic disorders and their inheritance can be autosomal dominant, recessive, X-linked or mitochondrial [2].Conversely, age-related hearing loss (presbycusis) or noiseinduced hearing loss (NIHL) is defined by a progressive course involving initially high frequencies and is considered multifactorial conditions with an environmental origin (i.e., vascular risk factors or noise exposure) [3].
Familial segregation and sequencing studies have been invaluable in developing our understanding of monogenic SNHL since a genetic component is present in ~ 50% of all hearing loss cases.However, the underlying molecular mechanisms of acquired SNHL remain poorly elucidated [4].While genome-wide association studies (GWAS) in adults affected by hearing loss continue to discover new candidate genes for hearing loss, a number of limitations to this approach have been identified.For example, while a genetic susceptibility may be highly relevant in specific types of hearing loss, it may not be the predominant factor for other types of hearing loss.It is challenging to make conclusions regarding etiological heterogeneities that encompass these large cohorts of self-reported hearing loss patients.Secondly, elucidating genomic mechanisms from the association in GWAS have proven difficult in past studies [4].Developing a strategy that allows for elucidation of molecular mechanisms of hearing loss in its various etiologies is crucial to the development of effective treatment strategies.
Emerging evidence is suggesting that DNA methylation may also have an important regulatory role in hearing loss and its associated conditions [5].DNA methylation is an epigenetic modification where a cytosine residue is converted to 5-methylcytosine (5mC) by DNA methyltransferases (DNMTs).Although the majority of methylation in human somatic cells is observed within a CpG dinucleotide context (within ~ 70% of gene promoters), it has also been identified within CpA, CpC and CpT contexts collectively known as non-CpG methylation [6].Both CpG and non-CpG methylation can silence gene expression by preventing transcription factor binding or through the recruitment of repressive complexes [6].Hence DNA methylation can lead to phenotypic changes without altering the underlying DNA sequence.
This systematic review aims to consolidate current literature linking DNA methylation and hearing loss in order to highlight remaining gaps in knowledge which may help elucidate a fuller comprehension of epigenetic changes in common and rare disorders associated with hearing loss.Understanding the precise mechanisms and specific genes involved in hearing function, which may be regulated through DNA methylation, could lead to the development of more refined studies that can help produce new therapeutic strategies for preventing or treating hearing loss and its associated conditions.

Study design
This review followed the PRISMA guidelines (Preferred Reported Items for Systematic Reviews and Meta-Analyses) [7] and adhered to the MOOSE checklist (Metaanalyses Of Observational Studies in Epidemiology) [8].
The review protocol was also registered on PROSPERO (CRD42023440491).
According to the methodology established for systematic reviews, the PICO question included the following items:

Search strategy
The search, conducted on November 15, 2023, used Pub-Med, Scopus and Cochrane databases with the following MesH terms: (hearing loss OR age-related hearing loss) AND (Cytosine OR methylation OR Epigenetics OR Epigenomics), and it was limited to original articles, published from the year 2000 onward.Replicates in references were removed, and articles incongruent with the review's objectives were omitted through the screening of their titles and abstracts.This process resulted in the retention of solely those records that conformed to the predefined inclusion criteria.In addition, the following exclusion criteria were used: • Studies that did not include any audiological assessments.• Studies published in other languages than English.
• Single-case reports, except multicase family or twin studies.

Data collection
Two different reviewers (V.P, P.P-C) independently extracted study characteristics and outcomes from all the included studies, and data were compared.A third reviewer (J.A.L.E) was consulted when a consensus could not be reached.Data pertaining to the review's objective were extracted from each article.From each study, the data collected included reference information (author and year of publication), geographical location, study design, research objectives, sample size, gender distribution, average age and the primary findings for each study (differentially methylated regions, DMR or genes, DMG).

Data synthesis/summary
We compiled the DMR and DMG across different studies for each condition or disease associated with hearing loss.We also summarized the studies involving mutations in the DNMT1 gene.

Analysis of subsets/subgroup
Studies were further subgrouped into three categories, (i) human studies of hearing loss and methylation, (ii) animal studies of hearing loss and methylation, (iii) hearing loss and DNMT1 mutations.All studies encompassed standardized audiometric testing for hearing loss in humans; auditory brainstem response in animals OR had a confirmed diagnosis of a disease where hearing loss is essential to pathophysiology.

Quality and risk of bias assessment
Was also evaluated; the ROBINS-E tool was used in nonrandomized Studies of Exposures [9].These tools consist of seven domains, namely: (1) confounding-induced bias, (2) bias in exposure measurement, (3) bias in participant selection for the study, (4) bias resulting from post-exposure interventions, (5) bias due to missing data, (6) bias in outcome measurement and (7) bias in the selection of reported results.Notably, domain 4 was deemed irrelevant for this review and was consequently excluded.The assessed risk of bias varied from "Low" to "Moderate," "High" or "Very High."Overall bias risk was determined by evaluating all domains collectively.A color-coded scale (white for not applicable, green for Low risk, yellow for Moderate risk, red for High risk and black for Very High risk) was employed to present a concise summary, as detailed in Table S1.The SYRCLE's risk of bias tool was used to assess animal studies [10] and included in Table S2.This tool contains 10 entries, which are related to 6 types of bias (selection bias, performance bias, detection bias, attrition bias, reporting bias and other biases), and helps to define the level of risk of bias based on several specific question for each domain.According to this, the risk of bias has been stablished as low/high/unclear.

Results
We selected a total of 25 articles which fit the inclusion criteria, 12 human DNA methylation studies, 5 experimental animal studies in mice [3] and rats [2] and 8 studies reporting mutations in the DNMT1 gene.Figure 1 details the flowchart for selection of the included articles.
Of the 12 human hearing loss studies, one study was conducted in relation to environmental exposure to Pb and Cd in children, whereas 11/12 were conducted in patients with concurrent presence of a relevant pathology namely, age-related hearing loss (ARHL, 6 studies), otosclerosis (OTSC), ototoxicity, Meniere's disease (MD) and diabetic-related hearing loss (DRHL) in a case-control setting (Tables 1, 2).Only one out of all human hearing loss studies (1/12) included a replication cohort in their study design.
The methodological approach for these studies fit into two main categories, site-specific methylation and genome-wide methylation.Some studies (4/11) investigated methylation variation in pre-defined sites with quantitative methylation-specific PCR.These studies revealed TNFSF11, CDH23 and SLC26A4 genes specifically have significant variation in methylation within gene encoding regions that is associated with audiologically tested variation in hearing loss.Other studies (7/11) performed whole-genome methylation array, reduced representation bisulfite sequencing (RRBS) or whole-genome bisulfite sequencing (WGBS).These studies identified significant variation in DNA methylation within gene promoter regions including DUSP4, C21orf58, ALG10, C3, LCK, GBX2.However, female-only studies highlighted a different subset of genes to be significantly differentially methylated, namely, TCF25, FGFR1, POLE, P2RX2, KCNQ5, ERBB3 and SOCS3.One study reported the significant differential methylation was detected in genes that were related to the concurrent disease, Type 2 Diabetes Mellitus, with no hearing loss genes being affected [11].
All animal studies, summarized in Table 3, were conducted in China on adult mice or rats except for one study which focused on rat offspring.The methodological approach for these studies fit into two main categories, site-specific methylation studies and histology paired with immunofluorescence.Site-specific DNA methylation assays (2/4) identified promoter hypermethylation of gjb2 gene in rats with inner hair cell damage induced by hypoxia.Immunofluorescence studies (2/4) independently showed that, in mice, inhibiting the DNA (cytosine-5)-methyltransferase 1 (dnmt1) enzyme can improve noise-induced hearing loss and promote hair cell regeneration.
The selection criteria additionally identified 8 human studies, where mutations in the DNMT1 gene were investigated in relation to hearing loss (Table 4).Methodological approaches for these studies were genotyping or exome sequencing.However, 2/8 of these studies included an additional DNA methylation assay in conjunction.In particular, 4/8 studies independently confirmed functional mutations in the DNMT1 gene to be strongly associated with autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN) with 3/8 of these studies consistently showing mutations within exon 21 of DNMT1 to be found in ADCA-DN patients or children of ADCA-DN patients.One study further highlighted 82 significantly hypermethylated regions in ADCA-DN patients with exon 21 DNMT1 mutations; however, it was concluded that further work with a more robust dataset would be needed to evaluate the importance of these hypermethylated regions to hearing loss.Patients with hereditary sensory and autonomic neuropathy (HSAN1), noise-induced hearing loss (NIHL), dementia and cognitive decline have all been identified to carry DNMT1 mutations within various locations.While 6/8 studies had a small sample size of n = 6 or less, only two cohort studies have been identified in the search.The largest cohort study (n = 1053) conducted in Chinese adults showed polymorphisms in both DNMT1 and DNMT3A that were implicated in noise-induced hearing loss (NIHL).

Risk of bias analysis
For human studies, the detailed analysis based on the seven domains of ROBINS-E is summarized in Table S1.According to this, 11 studies had a low risk of bias [11][12][13][14], 5 studies had a moderate risk of bias within at least one domain [15][16][17][18][19], and 4 studies were evaluated to have a high risk of bias within at least one domain [20][21][22][23].Animals studies risk of bias analysis is summarized in Table S2.

Discussion
This review was aimed to summarize emerging evidence which suggests DNA methylation may play an important role in a variety of conditions that are associated with hearing loss.We conducted a systematic review of all available literature where DNA methylation was investigated in conjunction with audiological testing in the context of aging as well as pathologies where hearing loss is a major aspect of the disease.We included a total of 25 studies, 12 performed in patients with concurrent presence of a relevant pathology (Tables 1, 2), 5 conducted in induced hearing loss animal models (Table 2) and 8 which focused on genetic screening of DNMT1 specifically (Table 3).Overall these studies showcase an association between DNA methylation and hearing loss with a strong need for larger, more robust datasets that may aid in developing a fuller understanding of the molecular mechanisms and key gene pathways that encompass hearing loss.
ARHL is a complex disorder resulting from the interaction of common and rare genetic variation with environmental exposure.Aging is associated with the additive effect of lifestyle and environmental factors both of which can be influenced by DNA methylation.In addition, there is already a large body of evidence which showcases the importance of DNA methylation in aging [24].This may explain why our search criteria found most methylation studies in humans (6/12) have been performed in individuals with ARHL (Table 1).Despite our selection criteria including 50% of studies being ARHL focused, only one of these studies had partially replicated their findings in an independent cohort (n = 203) [22].The lack of replication is an important consideration for future study designs since the potential clinical relevance of site-specific or global alterations in DNA methylation cannot be correlated to relevant hearing loss contexts without the added evidence of replication cohorts.Hence, at present there is strong association between variation in promoter methylation of genes such as CDH23, SLC26A4, TCF25 and POLE in women with ARHL; however, further investigations in larger cohorts and replication experiments are needed to consolidate these findings.In addition, genderspecific considerations are especially important in DNA methylation studies.Different DNA methylation patterns have been identified across several tissues in men compared to women.These differences have been attributed to mirror gender-specific transcriptomic and proteomic profiles [25][26][27].For example, comprehensive description of sex differences in DNA methylation changes with respect to aging in a whole blood dataset consisting of over 400 healthy subjects identified a number of regions where age-related increase in methylation variability was 15 times higher in males compared to females [28].
Hence future studies may benefit greatly by accounting for gender-specific studies.Furthermore, although there is substantial evidence linking aging and DNA methylation, whether there is a relationship between onset of hearing loss and DNA methylation remains largely under explored.In future investigation related to understanding whether DNA methylation across hearing loss genes may contribute to loss of function or missense variation would be beneficial (Table 2).Secondly, a wide variety of genome-wide DNA methylation assays have been utilized in the studies which were identified in our selection criteria.The main difference in these techniques, namely, RRBS, 27 K, 450 K, 850 K methylation arrays as well as WGBS, is the scope of genes and relevant genomic regions that can be assayed for differential methylation patterns simultaneously.While 450 K methylation array encompasses a wider range of genes compared to 27 K methylation arrays, RRBS includes all genes but only if they have CpG-rich regions as opposed to WGBS which encompasses all cytosine residues across the entire genome.This makes cross-comparisons between studies difficult since analysis strategies vary greatly for data acquired from various upstream assays.For example, P2RX2, KCNQ5, ERBB3 and SOCS3 genes were identified as having significant differential methylation in an ARHL female cohort in Tunisia [20] where RRBS was performed.However, significant differential methylation was identified within a different set of genes, TCF25, FGFR1, POLE, in a female only cohort of ARHL in the UK where a 27 K methylation array was used.This same study, from the TwinsUK registry, then confirmed differential methylation profiles in promoters of TCF25 and POLE in a second cohort using a 450 K methylation array [22].While both studies were focused on ARHL, due to inconsistency in study design, it is difficult to formulate the impacts of DNA methylation on ARHL in the context of ethnicity and environment.The genes identified in this study, TNC25 and POLE, have been implicated previously in hearing loss.However, a follow-up study assessing abnormalities in mRNA/protein expression in relevant hearing loss cohorts using the same sample material would have further consolidated these findings.Hence more comprehensive studies are necessary to develop our understanding of the impact of DNA methylation in hearing loss.Therefore, more cohort studies which encompass a standardized study design will aid immensely in growing our understanding of the potential role of DNA methylation in hearing loss as well as whether these changes are gender specific.
Nevertheless, reports of significant differential methylation in TCF25 and POLE genes are interesting findings due to the localization of the proteins they encode.TCF25 is a transcription factor, member of the ribosomeassociated quality control complex, comprising TCF25, LTN1 and NEMF genes; this complex is able to identify protein products from unproductive translation events, targeting them for degradation [29].The gene is widely expressed in the mouse cochlear epithelium in both sensory and supporting cells [30].POLE encodes a core catalytic subunit of DNA polymerase epsilon, involved in DNA repair and chromosomal DNA replication.Conversely to TCF25, RNAseq data from mice indicate that POLE is restricted to cochlear hair cells, particularly during development [31] Since differential DNA methylation patterns are known to affect gene expression patterns especially when present at gene promoter regions, further investigation is warranted to see how molecular mechanisms may be impacted in hearing loss through differential methylation.
The role of DNA methylation on rare diseases such as monogenic forms of sensorineural hearing loss (SNHL) has been seldom studied, the only exception being mutations in the DNMT1 gene, that it is associated with ADCA-DN syndrome.Since functional mutations in DNMT1 have been identified in patients with ADCA-DN, this provides a strong premise for further assessing global DNA methylation patterns in these patients.Only one of these studies further investigated global DNA methylation patterns [23].The study concluded further work with a more robust dataset is needed to make conclusive remarks.In addition, 450 K is an older assay with advancements such as 850 K methylation arrays as well as WGBS now more readily available than before.Hence a combination of replication cohorts, larger datasets with more robust methodologies has the potential to greatly improve our understanding of the possible role and related molecular mechanisms of DNA methylation in rare diseases such as monogenic forms of SNHL.
Several research areas remain unexplored in hearing loss methylation studies, such as non-CpG methylation [6].Non-CpG methylation has recently been attributed to allowing evolution of higher complexity in brain function for vertebrate species [32].Furthermore, the largest cohort study (n = 1053) included in our review which investigated NIHL, found polymorphisms in both DNMT1 and DNMT3A to be significant in their cohort [33].DNMT3A has an emerging role for instigating non-CpG methylation on the genome during brain development [34].Since hearing loss conditions are often associated with pathologies which can lead to cognitive decline, it may be a useful strategy to consider whether non-CpG methylation may be involved in certain hearing loss conditions.
From the 12 human studies, although many did not account for underlying genetic variation within their respective cohorts, 2/12 studies investigated whether specific genetic variants may be linked to an altered DNA methylation status.Both studies highlighted an association between the presence of specific polymorphisms with differential DNA methylation and subsequent differential gene expression.A recent study has identified 11.2 million unique SNP-CpG associations in peripheral blood taken from 3799 Europeans and 3,195 South Asian samples.The study presented strong evidence regarding the genetic regulation of DNA methylation [35].Hence studies which account for genetic variation in patients with confirmed hearing loss would be beneficial in the future.
In future it will also be useful to stratify subjects into high-or low-frequency hearing loss subsets.At present, not all study designs address this as an additional layer of complexity which may impact DNA methylation patterns detected.
This discussion is limited to studies which included audiometric assessment in their study design.Although this is an important criterion for assessing relationships between DNA methylation and hearing loss, we cannot ignore that studies which may have addressed this question from a different perspective may also contribute insightful findings which were beyond the scope of this review.

Conclusions
Overall, the literature collectively provides some evidence, suggesting variation in DNA methylation may play an important role in hearing loss, particularly in ARHL.Hearing ability is associated with methylation profile in the promoter of TCF25 and POLE genes in ARHI.
Epigenetic research should produce larger, more robust datasets where global DNA methylation patterns are investigated thoroughly within the context of standardized study designs.Furthermore, gender-specific crossstudy comparisons are needed for insightful knowledge on the role of DNA methylation in hearing loss processes.

Fig. 1
Fig. 1 Flow diagram for the DNA methylation study selection

Table 1
Descriptive features of selected studies which investigated DNA methylation in relation to hearing loss.All studies contain an element of confirmed diagnosis of hearing loss or audiometric tests

Table 2
Descriptive features of human studies highlighting the gene symbols and gene regions where differential DNA methylation was observed as well as definitions of hearing loss used for each study

Table 3
Descriptive features of selected studies which investigated DNA methylation in relation to hearing loss in animal models

Table 4
Descriptive features of selected studies which investigated DNMT1 mutations in humans with relation to hearing loss